一种用于丙型肝炎病毒丝氨酸蛋白酶的连续分光光度测定法。

A continuous spectrophotometric assay for the hepatitis C virus serine protease.

作者信息

Zhang R, Beyer B M, Durkin J, Ingram R, Njoroge F G, Windsor W T, Malcolm B A

机构信息

Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.

出版信息

Anal Biochem. 1999 Jun 1;270(2):268-75. doi: 10.1006/abio.1999.4109.

DOI:10.1006/abio.1999.4109

PMID:10334844

Abstract

The hepatitis C virus (HCV) encodes a chymotrypsin-like serine protease responsible for the processing of HCV nonstructural proteins and which is a promising target for antiviral intervention. Its relatively low catalytic efficiency has made standard approaches to continuous assay development only modestly successful. In this report, four continuous spectrophotometric substrates suitable for both high-throughput screening and detailed kinetic analysis are described. One of these substrates, Ac-DTEDVVP(Nva)-O-4-phenylazophenyl ester, is hydrolyzed by HCV protease with a second-order rate constant (kcat/Km) of 80,000 +/- 10,000 M-1 s-1. Together with its negligible rate of nonenzymatic hydrolysis under assay conditions (0.01 h-1), analysis of as little as 2 nM protease can be completed in under 10 min.

摘要

丙型肝炎病毒（HCV）编码一种类胰凝乳蛋白酶样丝氨酸蛋白酶，该酶负责HCV非结构蛋白的加工，是抗病毒干预的一个有前景的靶点。其相对较低的催化效率使得开发连续检测的标准方法仅取得了一定程度的成功。在本报告中，描述了四种适用于高通量筛选和详细动力学分析的连续分光光度法底物。其中一种底物，Ac-DTEDVVP（Nva）-O-4-苯基偶氮苯酯，被HCV蛋白酶水解，二级速率常数（kcat/Km）为80,000 +/- 10,000 M-1 s-1。在检测条件下其非酶促水解速率可忽略不计（0.01 h-1），在10分钟内即可完成低至2 nM蛋白酶的分析。